Aqueous two-phase extraction and characterization of thermotolerant alkaliphilic Cladophora hutchinsiae xylanase: biochemical properties and potential applications in fruit juice clarification and fish feed supplementation.

Xylanase finds extensive applications in diverse biotechnological fields such as biofuel production, pulp and paper industry, baking and brewing industry, food and feed industry, and deinking of waste paper. Here, polyethylene glycol (PEG)-phosphate aqueous two-phase system (ATPS) was applied for the purification of an alkaline active and thermotolerant xylanase from a marine source, Cladophora hutchinsiae (C. hutchinsiae). In the purification process, the effects of some experimental factors such as PEG concentration and PEG molar mass, potassium phosphate(K2HP04) concentration, and pH on xylanase distribution were systematically investigated. Relative enzymatic activity and purification factor obtained were 93.21% and 7.18, respectively. A single protein band of 28 kDa was observed on SDS-PAGE. The optimum temperature and pH of xylanase with beechwood xylan were 30 °C and 9.0, respectively. The Lineweaver-Burk graph was utilized to determine the Km (4.5 ± 0.8 mg/mL), Vmax (0.04 ± 0.01 U) and kcat (0.001 s-1) values of the enzyme. It was observed that the purified xylanase maintained 70% of its activity at 4 °C and was found stable at pH 4.0 by retaining almost all of its activity. Enzymatic activity was slightly enhanced with Na+, K+, Ca2+ and acetone. The highest increase in the reducing sugar amount was 53.6 ± 3.8, for orange juice at 50 U/mL enzyme concentration.

Acetylation of homogalacturonan and rhamnogalacturonan-I is catalyzed by a suite of trichome birefringence-like proteins.

Plant cell wall polysaccharides, including xylan, mannan, xyloglucan, and pectins, are often acetylated and members of the domain of unknown function 231 (DUF231)/trichome birefringence-like (TBL) family have been shown to be O-acetyltransferases mediating the acetylation of xylan, mannan, and xyloglucan. However, little is known about the O-acetyltransferases responsible for pectin acetylation. In this report, we biochemically characterized a suite of Arabidopsis DUF231/TBL proteins for their roles in pectin acetylation. We generated 24 TBL recombinant proteins in mammalian cells and demonstrated that 10 of them were able to transfer acetyl groups from acetyl-CoA onto the pectins homogalacturonan (HG) or rhamnogalacturonan-I (RG-I), and thus were named pectin O-acetyltransferase 1 to 10 (POAT1 to 10). It was found that POAT2,4,9,10 specifically acetylated HG and POAT5,6 acetylated RG-I, whereas POAT1,3,7,8 could act on both HG and RG-I. The acetylation of HG and RG-I by POATs was further corroborated by hydrolysis with pectin acetylesterases and by nuclear magnetic resonance spectroscopy. In addition, mutations of the conserved GDS and DXXH motifs in POAT3 and POAT8 were shown to lead to a loss of their ability to acetylate HG and RG-I. Furthermore, simultaneous RNA interference downregulation of POAT1,3,6,7,8 resulted in reduced cell expansion, impaired plant growth, and decreased pectin acetylation. Together, our findings indicate that these POATs are pectin O-acetyltransferases involved in acetylation of the pectin polysaccharides HG and RG-I.

The biosynthesis, degradation, and function of cell wall ß-xylosylated xyloglucan mirrors that of arabinoxyloglucan.

Xyloglucan is an abundant polysaccharide in many primary cell walls and in the human diet. Decoration of its α-xylosyl sidechains with further sugars is critical for plant growth, even though the sugars themselves vary considerably between species. Plants in the Ericales order - prevalent in human diets - exhibit ß1,2-linked xylosyl decorations. The biosynthetic enzymes responsible for adding these xylosyl decorations, as well as the hydrolases that remove them in the human gut, are unidentified. GT47 xyloglucan glycosyltransferase candidates were expressed in Arabidopsis and endo-xyloglucanase products from transgenic wall material were analysed by electrophoresis, mass spectrometry, and nuclear magnetic resonance (NMR) spectroscopy. The activities of gut bacterial hydrolases BoGH43A and BoGH43B on synthetic glycosides and xyloglucan oligosaccharides were measured by colorimetry and electrophoresis. CcXBT1 is a xyloglucan ß-xylosyltransferase from coffee that can modify Arabidopsis xyloglucan and restore the growth of galactosyltransferase mutants. Related VmXST1 is a weakly active xyloglucan α-arabinofuranosyltransferase from cranberry. BoGH43A hydrolyses both α-arabinofuranosylated and ß-xylosylated oligosaccharides. CcXBT1's presence in coffee and BoGH43A's promiscuity suggest that ß-xylosylated xyloglucan is not only more widespread than thought, but might also nourish beneficial gut bacteria. The evolutionary instability of transferase specificity and lack of hydrolase specificity hint that, to enzymes, xylosides and arabinofuranosides are closely resemblant.

Xylan clustering on the pollen surface is required for exine patterning.

Xylan is a crosslinking polymer that plays an important role in the assembly of heterogeneous cell wall structures in plants. The pollen wall, a specialized cell wall matrix, exhibits diverse sculpted patterns that serve to protect male gametophytes and facilitate pollination during plant reproduction. However, whether xylan is precisely anchored into clusters and its influence on pollen wall patterning remain unclear. Here, we report xylan clustering on the mature pollen surface in different plant species that is indispensable for the formation of sculpted exine patterns in dicot and monocot plants. Chemical composition analyses revealed that xylan is generally present at low abundance in the mature pollen of flowering plants and shows plentiful variations in terms of substitutions and modifications. Consistent with the expression profiles of their encoding genes, genetic characterization revealed IRREGULAR XYLEM10-LIKE (IRX10L) and its homologous proteins in the GT47 family of glycosyltransferases as key players in the formation of these xylan micro-/nano-compartments on the pollen surface in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa). A deficiency in xylan biosynthesis abolished exine patterning on pollen and compromised male fertility. Therefore, our study outlines a mechanism of exine patterning and provides a tool for manipulating male fertility in crop breeding.

Immunochemical Identification of the Main Cell Wall Polysaccharides of the Early Land Plant Marchantia polymorpha.

Plant primary cell walls are composite structures surrounding the protoplast and containing pectins, hemicelluloses, and cellulose polysaccharides, as well as proteins. Their composition changed during the evolution of the green lineage from algae to terrestrial plants, i.e., from an aquatic to a terrestrial environment. The constraints of life in terrestrial environments have generated new requirements for the organisms, necessitating adaptations, such as cell wall modifications. We have studied the cell wall polysaccharide composition of thalli of Marchantia polymorpha, a bryophyte belonging to one of the first land plant genera. Using a collection of specific antibodies raised against different cell wall polysaccharide epitopes, we were able to identify in polysaccharide-enriched fractions: pectins, including low-methylesterified homogalacturonans; rhamnogalacturonan I with arabinan side-chains; and hemicelluloses, such as xyloglucans with XXLG and XXXG modules, mannans, including galactomannans, and xylans. We could also show the even distribution of XXLG xyloglucans and galactomannans in the cell walls of thalli by immunocytochemistry. These results are discussed with regard to the cell wall proteome composition and in the context of the evolution of the green lineage. The cell wall polysaccharides of M. polymorpha illustrate the transition from the charophyte ancestors of terrestrial plants containing xyloglucans, xylans and mannans as hemicelluloses, and embryophytes which do not exhibit mannans as major primary cell wall polysaccharides.

The effect of different wheat varieties and exogenous xylanase on bird performance and utilization of energy and nutrients.

The aims of the present study were to first, determine the xylan fractions of 10 different wheat cultivar samples and their response to treatment by the same commercial xylanase enzyme preparation. Second, use information obtained to select 5 of the wheats for use within a feeding experiment to determine whether the rate of xylan release can be used to predict the feeding value of the wheats when diets have been supplemented with xylanase. Treatment of 10 different wheat varieties by the same enzyme resulted in varying levels of hydrolysis. Soluble xylan content ranged from 7.85 to 14.40 and 3.20 to 5.13 (mg/g) when treated with and without xylanase, respectively. Oligosaccharide content ranged from 0.34 to 1.58 and 0.05 to 0.54 (mg/g) when treated with and without xylanase, respectively. Five of the 10 wheats were then selected based on the determined xylan fractions to use within a feeding experiment. A total of 360 male Ross 308 broilers were randomly allocated to 60 raised floor pens. A soybean meal (SBM) balancer feed was formulated to contain 12.07 MJ/kg apparent metabolizable energy (AME) and 392.9 g/kg crude protein (CP). Five diets were prepared by mixing 630 g/kg of each of the 5 experimental wheats with 370 g/kg of the balancer. Each diet was split into 2, one of which was supplemented with 100 g/MT of Econase XT (223,000 BXU/g), resulting in a total of 10 diets. The birds were fed the diets from 0 to 28 d of age. Wheat cultivar had an effect (P = 0.044) on feed intake (FI), while the addition of xylanase increased (P < 0.05) weight gain (WG) and improved feed conversion ratio (FCR). Various interactions were observed (P < 0.05) between wheat cultivars and xylanase for AME and nutrient utilization. This study suggests that wheats treated with the same xylanase, differ in their susceptibility to release soluble xylan and oligosaccharides, which may partially explain the varying performance and nutrient digestibility responses noted in the literature.

Impact of fermentable fiber, xylo-oligosaccharides and xylanase on laying hen productive performance and nutrient utilization.

This study evaluated the impact of feeding xylo-oligosaccharides (XOS), fermentable fiber in the form of wheat bran (WB), and xylanase (XYL) on laying hen productive performance and nutrient digestibility. The hypothesis was that the WB would provide the microbiota in the hindgut with fermentable dietary xylan, and the XOS and XYL would further upregulate xylan fermentation pathways, resulting in improved nutrient utilization. Isa Brown hens (n = 96) were obtained at 39 wk of age. They were fed 12 dietary treatments, 8 hens per treatment, for 56 d. A commercial laying hen ration was fed, and for half of the treatments 10% of this ration was directly replaced with WB. The diets were then supplemented with either 1) no supplements; 2) XOS 50 g/t; 3) XOS 2000 g/t; 4) XYL (16,000 BXU/kg); 5) XYL + XOS 50 g/t, or 6) XYL + XOS 2,000 g/t. Hen performance and egg quality were measured every 14 d. On d56, ileum digesta samples were collected for determination of starch, nonstarch polysaccharide (NSP), XOS, protein, energy, and starch digestibility. Ceca digesta samples were also collected for analysis of XOS, short chain fatty acid (SCFA), xylanase and cellulase activity and microbial counts. Feeding 2,000 g/t XOS increased ileal protein digestibility. Combined 2,000 g/t XOS and XYL increased cecal Bifidobacteria concentration. This combination also increased cecal xylanase activity in birds fed the control diet. Cecal cellulase activity was improved by feeding WB, XYL, and 2,000 g/t XOS. XYL increased cecal lactate production. Feeding 2,000 g/t XOS with WB increased insoluble NSP degradability and shell breaking strength at d56. In summary, supplementing laying hen diets with fermentable fiber, XYL and XOS increases utilization of dietary xylan, improving nutrient utilization, performance, and gastrointestinal health.

Metagenomic Analysis of in Vitro Ruminal Fermentation Reveals the Role of the Copresent Microbiome in Plant Biomass Degradation.

In vitro ruminal fermentation is considered an efficient way to degrade crop residue. To better understand the microbial communities and their functions during in vitro ruminal fermentation, the microbiome and short chain fatty acid (SCFA) production were investigated using the metagenomic sequencing and rumen simulation technique (RUSITEC) system. A total of 1677 metagenome-assembled genomes (MAGs) were reconstructed, and 298 MAGs were found copresenting in metagenomic data of the current work and 58 previously ruminal representative samples. Additionally, the domains related to pectin and xylan degradation were overrepresented in the copresent MAGs compared with total MAGs. Among the copresent MAGs, we obtained 14 MAGs with SCFA-synthesis-related genes positively correlated with SCFA concentrations. The MAGs obtained from this study enable a better understanding of dominant microbial communities across in vivo and in vitro ruminal fermentation and show promise for pointing out directions for further research on in vitro ruminal fermentation.

Structural variation and spatial polysaccharide profiling of intervessel pit membranes in grapevine.

BACKGROUND AND AIMS: Intervessel pit membranes (PMs) are important cell wall structures in the vessel system that may impact a plant's water transport and its susceptibility to vascular diseases. Functional roles of intervessel PMs largely depend on their structure and polysaccharide composition, which are the targets of this study. METHODS: With grapevine used as a model plant, this study applied an immunogold-scanning electron microscopy technique to simultaneously analyse at high resolution intervessel PM structures and major pectic and hemicellulosic polysaccharides that make up intervessel PMs. KEY RESULTS: Intervessel PMs in functional xylem showed significant structural variation, with about 90 % of them being structurally intact with smooth or relatively smooth surfaces and the remaining 10 % with progressively degraded structures. The results also elucidated details of the removal process of cell wall materials from the intervessel PM surface toward its depth during its natural degradation. Four groups of pectic and hemicellulosic polysaccharides were immunolocalized in intervessel PMs and differed in their spatial distribution and abundance. Weakly methyl-esterified homogalacturonans (WMe-HGs, detected by JIM5) were abundant in the surface layer, heavily methyl-esterified homogalacturonans (HMe-HGs, detected by JIM7) and xylans detected by CCRC-M140 were mostly found in deeper layers, and fucosylated xyloglucans (F-XyGs, detected by CCRC-M1) were more uniformly distributed at different depths of the intervessel PM. CONCLUSIONS: Intervessel PMs displayed diverse structural variations in grapevine. They contained certain major groups of pectic and hemicellulosic polysaccharides with different spatial distributions and abundance. This information is crucial to reveal the polysaccharide profiling of the primary cell wall and to understand the roles of intervessel PMs in the regulation of water transport as well as in a plant's susceptibility to vascular diseases.

Building an extensible cell wall.

This article recounts, from my perspective of four decades in this field, evolving paradigms of primary cell wall structure and the mechanism of surface enlargement of growing cell walls. Updates of the structures, physical interactions, and roles of cellulose, xyloglucan, and pectins are presented. This leads to an example of how a conceptual depiction of wall structure can be translated into an explicit quantitative model based on molecular dynamics methods. Comparison of the model's mechanical behavior with experimental results provides insights into the molecular basis of complex mechanical behaviors of primary cell wall and uncovers the dominant role of cellulose-cellulose interactions in forming a strong yet extensible network.

Dynamics of cell wall polysaccharides during the elongation growth of rye primary roots.

MAIN CONCLUSION: In cells of growing rye roots, xyloglucans and homogalacturonans demonstrate developmental stage specificity, while different xylans have tissue specificity. Mannans, arabinans and galactans are also detected within the protoplast. Mannans form films on sections of fresh material. The primary cell walls of plants represent supramolecular exocellular structures that are mainly composed of polysaccharides. Cell wall properties and architecture differ between species and across tissues within a species. We revised the distribution of cell wall polysaccharides and their dynamics during elongation growth and histogenesis in rye roots using nonfixed material and the spectrum of antibodies. Rye is a member of the Poaceae family and thus has so-called type II primary cell walls, which are supposed to be low in pectins and xyloglucans and instead have arabinoxylans and mixed-linkage glucans. However, rye cell walls at the earliest stages of cell development were enriched with the epitopes of xyloglucans and homogalacturonans. Mixed-linkage glucan, which is often considered an elongation growth-specific polysaccharide in plants with type II cell walls, did not display such dynamics in rye roots. The cessation of elongation growth and even the emergence of root hairs were not accompanied by the disappearance of mixed-linkage glucans from cell walls. The diversity of xylan motifs recognized by different antibodies was minimal in the meristem zone of rye roots, but this diversity increased and showed tissue specificity during root growth. Antibodies specific for xyloglucans, galactans, arabinans and mannans bound the cell content. When rye root cells were cut, the epitopes of xyloglucans, galactans and arabinans remained within the cell content, while mannans developed net-like or film-like structures on the surface of sections.

The graft framework: Quantitative changes in cell wall matrix polysaccharides throughout the tomato graft union formation.

Plant cell walls provide essential functions in cell recognition, differentiation, adhesion and wound responses. Therefore, it is tempting to hypothesize that cell walls play a key role in grafting, but to date there are no quantitative studies targeting on cell wall changes during grafting. The aim of this work was to investigate the dynamics of pectic and hemicellulosic polysaccharides at the graft junctions in tomato homografts throughout the first 12 days after grafting. Cell wall fractionation, combined with ATR-FTIR spectroscopy and gas-chromatography, evidenced a marked increase in pectin content and a decrease in the degree of methyl-esterification of homogalacturonan in scion and rootstock throughout grafting. Also, recovery of tightly-bound hemicelluloses decreased at late times after grafting suggesting an increase of cross-linked hemicelluloses along grafting. In addition, immuno-dot assays revealed an increase in xyloglucan and arabinogalactan proteins in the first days after grafting, pointing to a presumed role in tissue adhesion-cohesion.

Surface-enhanced Raman spectroscopic chemical imaging reveals distribution of pectin and its co-localization with xyloglucan inside onion epidermal cell wall.

The primary plant cell wall is a complex matrix composed of interconnected polysaccharides including cellulose, hemicellulose, and pectin. Changes of this dynamic polysaccharide system play a critical role during plant cell development and differentiation. A better understanding of cell wall architectures can provide insight into the plant cell development. In this study, a Raman spectroscopic imaging approach was developed to visualize the distribution of plant cell wall polysaccharides. In this approach, Surface-enhanced Raman scattering (SERS through self-assembled silver nanoparticles) was combined with Raman labels (4-Aminothiophenol. 4ATP) and targeted enzymatic hydrolysis to improve the sensitivity, specificity, and throughput of the Raman imaging technique, and to reveal the distribution of pectin and its co-localization with xyloglucan inside onion epidermal cell (OEC) wall. This technique significantly decreased the required spectral acquisition time. The resulted Raman spectra showed a high Raman signal. The resulted Raman images successfully revealed and characterized the pectin distribution and its co-localization pattern with xyloglucan in OEC wall.

Effect of different carbon sources on the growth and enzyme production of a toxigenic and a non-toxigenic strain of Aspergillus flavus.

Two strains of A. flavus one toxigenic (CECT 2687) and the other non-toxigenic (NRRL 6541) were studied for their genomic potential, growth capacity, and the production of enzymes on simple sugars, polysaccharides, and complex substrates under solid-state fermentation (SSF). According to the genome analysis, this fungus has many genes to degrade different types of polysaccharides and therefore it would be able to grow on different substrates. Both strains grow in all the carbon sources, but visibly CECT2687 grows slower than NRRL6541. However, we propose the growth index (GI) to establish a dry weight-diameter relationship as a more reliable measure that truly shows the growth preferences of the fungus. Considering this, the NRRL6541 shows less growth in 11 of the 16 evaluated carbon sources than CECT2687. Complex substrates were the best carbon source for the growth of both strains. Corncob (CC) induced the production of xylanases, pectinases, and almost all the accessory enzymes evaluated (except for α-xylosidase) this could make it an agricultural waste of interest to produce hemicellulolytic enzymes. Both strains produce a great variety of xylanases and pectinases (pathogenicity factors) making A. flavus a good potential candidate for the degradation of polysaccharides with a high content of xylan and pectin.

Unraveling the xylanolytic potential of Acidobacteria bacterium AB60 from Cerrado soils.

The presence of genes for glycosyl hydrolases in many Acidobacteria genomes indicates an important role in the degradation of plant cell wall material. Acidobacteria bacterium AB60 was obtained from Cerrado oligotrophic soil in Brazil, where this phylum is abundant. The 16S rRNA gene analyses showed that AB60 was closely related to the genera Occallatibacter and Telmatobacter. However, AB60 grew on xylan as carbon source, which was not observed in Occallatibacter species; but growth was not detected on medium containing carboxymethyl cellulose, as observed in Telmatobacter. Nevertheless, the genome analysis of AB60 revealed genes for the enzymes involved in cellulose as well as xylan degradation. In addition to enzymes involved in xylan degradation, α-l-rhamnosidase was detected in the cultures of AB60. Functional screening of a small-insert genomic library did not identify any clones capable of carboxymethyl cellulose degradation, but open reading frames coding α-l-arabinofuranosidase and α-l-rhamnosidase were present in clones showing xylan degradation halos. Both enzymes act on the lateral chains of heteropolymers such as pectin and some hemicelluloses. These results indicate that the hydrolysis of α-linked sugars may offer a metabolic niche for slow-growing Acidobacteria, allowing them to co-exist with other plant-degrading microbes that hydrolyze ß-linked sugars from cellulose or hemicellulose backbones.

Xylanase supplementation in corn-based swine diets: a review with emphasis on potential mechanisms of action.

Corn is a common energy source in pig diets globally; when financially warranted, industrial corn coproducts, such as corn distiller's dried grains with solubles (DDGS), are also employed. The energy provided by corn stems largely from starch, with some contribution from protein, fat, and non-starch polysaccharides (NSP). When corn DDGS are used in the diet, it will reduce starch within the diet; increase dietary protein, fat, and NSP levels; and alter the source profile of dietary energy. Arabinoxylans (AXs) comprise the majority of NSP in corn and its coproducts. One strategy to mitigate the antinutritive effects of NSP and improve its contribution to energy is by including carbohydrases within the diet. Xylanase is a carbohydrase that targets the ß-1,4-glycosidic bonds of AX, releasing a mixture of smaller polysaccharides, oligosaccharides, and pentoses that could potentially be used by the pig. Xylanase is consistently effective in poultry production and moderately consistent in wheat-based swine diets, but its efficacy in corn-based swine diets is quite variable. Xylanase has been shown to improve the digestibility of various components of swine-based diets, but this seldom translates into an improvement in growth performance. Indeed, a review of xylanase literature conducted herein suggests that xylanase improves the digestibility of dietary fiber at least 50% of the time in pigs fed corn-based diets, but only 33% and 26% of the time was there an increase in average daily gain or feed efficiency, respectively. Intriguingly, there has been an abundance of reports proposing xylanase alters intestinal barrier integrity, inflammatory responses, oxidative status, and other health markers in the pig. Notably, xylanase has shown to reduce mortality in both high and low health commercial herds. These inconsistencies in performance metrics, and unexpected health benefits, warrant a greater understanding of the in vivo mechanism(s) of action (MOA) of xylanase. While the MOA of xylanase has been postulated considerably in the literature and widely studied in in vitro settings, in wheat-based diets, and in poultry, there is a dearth of understanding of the in vivo MOA in pigs fed corn-based diets. The purpose of this review is to explore the role of xylanase in corn-based swine diets, discuss responses observed when supplemented in diets containing corn-based fiber, suggest potential MOA of xylanase, and identify critical research gaps.

Another building block in the plant cell wall: Barley xyloglucan xyloglucosyl transferases link covalently xyloglucan and anionic oligosaccharides derived from pectin.

We report on the homo- and hetero-transglycosylation activities of the HvXET3 and HvXET4 xyloglucan xyloglucosyl transferases (XET; EC 2.4.1.207) from barley (Hordeum vulgare L.), and the visualisation of these activities in young barley roots using Alexa Fluor 488-labelled oligosaccharides. We discover that these isozymes catalyse the transglycosylation reactions with the chemically defined donor and acceptor substrates, specifically with the xyloglucan donor and the penta-galacturonide [α(1-4)GalAp]5 acceptor - the homogalacturonan (pectin) fragment. This activity is supported by 3D molecular models of HvXET3 and HvXET4 with the docked XXXG donor and [α(1-4)GalAp]5 acceptor substrates at the -4 to +5 subsites in the active sites. Comparative sequence analyses of barley isoforms and seed-localised TmXET6.3 from nasturtium (Tropaeolum majus L.) permitted the engineering of mutants of TmXET6.3 that could catalyse the hetero-transglycosylation reaction with the xyloglucan/[α(1-4)GalAp]5 substrate pair, while wild-type TmXET6.3 lacked this activity. Expression data obtained by real-time quantitative polymerase chain reaction of HvXET transcripts and a clustered heatmap of expression profiles of the gene family revealed that HvXET3 and HvXET6 co-expressed but did not share the monophyletic origin. Conversely, HvXET3 and HvXET4 shared this relationship, when we examined the evolutionary history of 419 glycoside hydrolase 16 family members, spanning monocots, eudicots and a basal Angiosperm. The discovered hetero-transglycosylation activity in HvXET3 and HvXET4 with the xyloglucan/[α(1-4)GalAp]5 substrate pair is discussed against the background of roles of xyloglucan-pectin heteropolymers and how they may participate in spatial patterns of cell wall formation and re-modelling, and affect the structural features of walls.

Formation of Cellulose-Based Composites with Hemicelluloses and Pectins Using Komagataeibacter Fermentation.

Komagataeibacter xylinus synthesizes cellulose in an analogous fashion to plants. Through fermentation of K. xylinus in media containing cell wall polysaccharides from the hemicellulose and/or pectin families, composites with cellulose can be produced. These serve as general models for the assembly, structure, and properties of plant cell walls. By studying structure/property relationships of cellulose composites, the effects of defined hemicellulose and/or pectin polysaccharide structures can be investigated. The macroscopic nature of the composites also allows composite mechanical properties to be characterized.The method for producing cellulose-based composites involves reviving and then culturing K. xylinus in the presence of desired hemicelluloses and/or pectins. Different conditions are required for construction of hemicellulose- and pectin-containing composites. Fermentation results in a floating mat or pellicle of cellulose-based composite that can be recovered, washed, and then studied under hydrated conditions without any need for intermediate drying.

Characterization of two family AA9 LPMOs from Aspergillus tamarii with distinct activities on xyloglucan reveals structural differences linked to cleavage specificity.

Aspergillus tamarii grows abundantly in naturally composting waste fibers of the textile industry and has a great potential in biomass decomposition. Amongst the key (hemi)cellulose-active enzymes in the secretomes of biomass-degrading fungi are the lytic polysaccharide monooxygenases (LPMOs). By catalyzing oxidative cleavage of glycoside bonds, LPMOs promote the activity of other lignocellulose-degrading enzymes. Here, we analyzed the catalytic potential of two of the seven AA9-type LPMOs that were detected in recently published transcriptome data for A. tamarii, namely AtAA9A and AtAA9B. Analysis of products generated from cellulose revealed that AtAA9A is a C4-oxidizing enzyme, whereas AtAA9B yielded a mixture of C1- and C4-oxidized products. AtAA9A was also active on cellopentaose and cellohexaose. Both enzymes also cleaved the ß-(1→4)-glucan backbone of tamarind xyloglucan, but with different cleavage patterns. AtAA9A cleaved the xyloglucan backbone only next to unsubstituted glucosyl units, whereas AtAA9B yielded product profiles indicating that it can cleave the xyloglucan backbone irrespective of substitutions. Building on these new results and on the expanding catalog of xyloglucan- and oligosaccharide-active AA9 LPMOs, we discuss possible structural properties that could underlie the observed functional differences. The results corroborate evidence that filamentous fungi have evolved AA9 LPMOs with distinct substrate specificities and regioselectivities, which likely have complementary functions during biomass degradation.

Elongating maize root: zone-specific combinations of polysaccharides from type I and type II primary cell walls.

The dynamics of cell wall polysaccharides may modulate the cell wall mechanics and thus control the expansion growth of plant cells. The unique composition of type II primary cell wall characteristic of grasses suggests that they employ specific mechanisms for cell enlargement. We characterized the transcriptomes in five zones along maize root, clustered the expression of genes for numerous glycosyltransferases and performed extensive immunohistochemical analysis to relate the changes in cell wall polysaccharides to critical stages of cell development in Poaceae. Specific patterns of cell wall formation differentiate the initiation, realization and cessation of elongation growth. Cell walls of meristem and early elongation zone represent a mixture of type I and type II specific polysaccharides. Xyloglucans and homogalacturonans are synthesized there actively together with mixed-linkage glucans and glucuronoarabinoxylans. Rhamnogalacturonans-I with the side-chains of branched 1,4-galactan and arabinan persisted in cell walls throughout the development. Thus, the machinery to generate the type I primary cell wall constituents is completely established and operates. The expression of glycosyltransferases responsible for mixed-linkage glucan and glucuronoarabinoxylan synthesis peaks at active or late elongation. These findings widen the number of jigsaw pieces which should be put together to solve the puzzle of grass cell growth.